This invention relates to copolymers containing N-Acetyl Glucosamine (NAG) of formula (1) having molecular weight ranging from 1,000 Daltons to 2,00,000 Daltons herein below 
wherein,
R is H, CH3, C2H5, C6H5; R1 is H, CH3, C2H5, C6H5; R2 is H, CH3, C2H5, C6H5;
X varies between 4-10; m is from 3 to 500; n is from 2 to 50; p is from 2 to 50; L is OH, NH2,OCH3, NHCH(CH3)2; and
Y may be N-Acetyl Glucosamine (NAG), mannose, galactose, sialic acid, fructose, ribulose, erythrolose, xylulose, psicose, sorbose, tagatose, glucopyranose, fructofuranose, deoxyribose, galactosamine, sucrose, lactose, isomaltose, maltose, cellobiose, cellulose and amylose.
More particularly it relates to the said copolymers containing carbohydrate ligands and preparation thereof mentioned herein. Still more particularly it relates to copolymers which bind more strongly to lysozyme than NAG itself.
The copolymers of the present invention as mentioned above are prepared by reacting monomer of formula (2) herein below with polymerizable macromer of formula (3) claimed in another copending. 
wherein,
R1 is H, CH3, C2H5, C6H5, L is OH, NH2, OCH3 and NHCH(CH3)2 
wherein,
R is H, CH3, C2H5, C6H5, R2 is H, CH3, C2H5, C6H5. 
X may be between 4-10, p is from 2 to 50.
Y may be N-Acetyl Glucosamine (NAG), mannose, galactose, sialic acid, fructose, ribulose, erythrolose, xylulose, psicose, sorbose, tagatose, glucopyranose, fructofuranose, deoxyribose, galactosamine, sucrose, lactose, isomaltose, maltose, cellobiose, cellulose and amylose.
The copolymers may be used for inhibition of viral infections and the recoveries of biomolecules. The approach of synthesis of copolymers with ligand N-Acetyl Glucosamine (NAG) is a generic and can be used for other ligands such as sialic acid, galactose and mannose.
Carbohydrates exhibit molecular diversity and wide structural variations, which makes carbohydrates alternative ligands for competitive binding to inhibit the infections. Sharon et al., (Science, 246:227-234,1989) reported carbohydrate portions of glyco-conjugate molecules to be an important entity in biology. One of the major advantage of carbohydrate modification may be that it can impart change in physical characteristics such as solubility, stability, activity, antibody recognition and susceptibility to enzyme.
Carbohydrates can be incorporated in polymer chain and can be utilized for binding to the receptors. Thereby, the polymers can be coupled with the other polymers containing ligands to impart multivalent effect.
Carbohydrates play a crucial role in biological phenomena and therefore such molecules have attracted the attention of chemists and biochemists. These biomolecules are ubiquitous, figuring prominently in various processes such as cell differentiation, cell growth, inflammation, viral and bacterial infection, tumorigenesis and metastasis (Rouhi A.,M., C and EN, September 23,62-66,1996).
Infections caused by bacteria and virus are a result of host receptor interactions. The foremost step for the infection is the adhesion of the ligands present on the infectious microbe to the receptors of the host cells. Adhesion and interactions have to be strong for a successful infection. If the adhesion is not adequate then normal defense mechanism can intercept this process. Viruses and bacteria for example interact with certain saccharides of the host cell. Bacteria express a large number of lectins and are used to adhere to glycocalyx of the host cell through a multivalent interactions. Agglutination of erythrocytes is a case in point.
Many alterations and modifications of the naturally occurring O/N-glycosidic sugars are being reported and is an area of prime interest to the chemist and biochemist. Carbohydrates are usually linked to other moieties such as lipids or proteins. Belvilacqua et al., (Science, 243:1160,1989) have demonstrated the role of carbohydrates along with proteins and nucleic acids as a primary biological information carriers.
Recently few reports have been published to justify the use of carbohydrates in therapeutics for human, as they can play crucial role in prevention of viral and bacterial infections. Krepinsky et al. (U.S. Pat. No. 6,184,368, 2001) suggested the application of carbohydrates in preventing the infections. Mandeville, et al. (U.S Pat. No. 5,891,862,1999) reported the use of polyvalent polymers containing carbohydrates for the treatment of rotavirus infection.
Polyvalent molecules bind to the receptor molecules through multiple contacts, which results in strong binding. However, the synthesis of ligands is critical and involves multiple steps. The polyvalent interactions can be maximized by incorporation of ligands optimally tailored based on the understanding of the binding between the ligand and the host receptor. The enhanced interactions are important especially when the ligands are expensive e.g. sialic acid.
The inventors of the present invention have also observed that interactions can be enhanced by 1) appropriate incorporation of the ligand 2) incorporation of spacer chain and 3) steric stabilization/exclusion using polymers.
Spaltenstein et al., (J.Am.Chem. Soc.,113:686,1991) reported increased interaction between the receptor and ligand due to plurality of binding ligands and the receptors on the host surface. This was illustrated by the influenza virus hemagglutinin, which binds to neuraminic acid on the cell surface, which has a greater affinity for its receptor when a polyvalent structure is presented.
Protein carbohydrate interactions are of low affinity. If relative density and spatial arrangement of ligands incorporated is optimized, then the binding can be substantially enhanced. The enhanced interaction between molecular conjugate with a specific binding site of biomolecule also finds applications in affinity separations, drug delivery and biotechnology.
Design of high affinity protein carbohydrate binding systems can provide an alternative strategy for the treatment of infectious diseases e.g. influenza and rotavirus. This has the advantage as such agents will not have pathogen resistance to antibiotics and drugs. A new approach to treat influenza is based on the principle of inhibition of virus to the host cells. The inhibitors like sialic acid anchored to polymeric or liposomal carriers have been reported in the past.
Since monovalent interactions of natural oligosaccharides are weak, they need to be used in large quantities for an effective treatment. This problem can be overcome by synthesizing polyvalent carbohydrate molecules (Zopf, D., Roth, S. Lancet 347, 1017, 1996). The concept is attractive since it would provide a non-toxic therapeutic to a wide range of human diseases. But synthesis of such compounds is critical and requires knowledge of the host-cell binding mechanism.
Polymeric ligands that bind to the virus more powerfully than the Red Blood Cells will prevent the influenza infection. Similar binding is also involved in rotavirus infections. (Mandeville, et al. U.S. Pat. No. 6,187,762, 2001)
Advantage of carbohydrate modification lies in that it may impart change in physical characteristics such as solubility, stability, activity, antibody recognition and susceptibility to enzyme.
Sigal, et al., demonstrated (J. Am. Chem. Soc. 118:16, 3789-3800,1996) haemagglutination prevention by saccharides multivalent glycoconjugates, which bind to the bacterial lectins and thus inhibit bacterial adhesion. Damschroder et al. (U.S. Pat. No. 2,548,520,1951) demonstrated high molecular weight preformed polymers conjugated with unsaturated monomers or proteins. Synthesis of high molecular weight materials of this kind generally requires temperatures up to 100xc2x0 C. Such high temperatures are not well tolerated by most of the proteins as they are thermolabile. Thus the methods described are unsuitable for producing polymers of biologically active molecules.
Carbohydrates can be used as functionalized ligands by incorporation into polymer backbone. The copolymers containing NAG and macromer containing polyvalent NAG form a multivalent conjugate.
Multivalent copolymers of varied length and density will be useful for receptor ligand interactions of biological origin. Various chemical and chemoenzymetic methods have been reported in the past for the preparation of di- and trivalent ligands, dendrimers, and high molecular weight polymers but have proven to be complicated to synthesize. Thus, there is necessity of a simple methodology to obtain multivalent ligands and polymers of varying chain length. Mammen,M., and Whitesides,G.,M., demonstrated that (J.Med.Chem.38:21,4179-90,1995) agglutination of erythrocytes caused by influenza virus could be prevented by use of polyvalent sialic acid inhibitor. Moreover, they suggested two favorable mechanisms for inhibition between the surfaces of virus and erythrocytes 1) high-affinity binding through polyvalency and 2) steric stabilization. This novel approach is a model for pathogen-host interactions.
Sigel, et al.(J.Am.Chem.Soc.118:16,3789-3800,1996) prepared polymers containing sialoside and evaluated the efficiency of inhibition of influenza virus in terms of inhibition constants (Ki).Although the authors observed that the extent of inhibition and minimum inhibition concentration decreased with increase in polymer molecular weight and sialic acid content, it was also noted that not all sialic acid ligands were involved in binding with the virus. This clearly indicates need of tailoring the polymer structure so that higher fraction of ligands is involved in binding.
Spevak et al. (J. Am. Chem. Soc., 115,1146-1147, 1993) reported the polymerized liposomes containing C-glycosides of sialic acid, which were potent inhibitors of influenza virus. Moreover the authors demonstrated that the infection was inhibited more effectively when the ligand bearing monomer was polymerized.
Various methods have been reported in the past to synthesize multivalent ligands such as ring-opening metathesis polymerization (ROMP). ROMP has been used to generate defined, biologically active polymers by Gibson et al., (Chem. Comm., 1095-1096,1997) and Biagini et al., (Polymer, 39, 1007-1014,1998).
Recent advancements in the field of glycoscience have demonstrated enhanced binding between carbohydrate ligands and specific receptors as a result of the cluster effect. These interactions are result from intrinsic properties of such ligands.
Various methods have been reported in the past for the synthesis of glycoconjugate oligomers and the clusters for the receptor binding activity. Nishimora, et al. (Macromolecules, 27, 4876-4880,1994) synthesized sugar homopolymer clusters from acrylamidoalkyl glycosides of N-Acetyl-D-Glucosamine. On addition of the polymer clusters, binding to WGA was enhanced.
Various methods for the preparation of random, block or graft copolymers containing N-isopropyl acrylamide monomer are reported in the past. e.g. a number of monomers including butyl methacrylate, N-isopropyl methacrylamide and dextran sulfate have been polymerized with N-isopropyl acrylamide to prepare random copolymers. Hoffman et al. (J. Biomater. Sci., Polym. Ed., 4(5), 545 (1993) synthesized carboxyl terminated poly(N-isopropyl acrylamide) oligomers, which were reacted with biopolymers to form thermo-reversible polymer-enzyme conjugates.
Therefore, the objective of the present work is to synthesize copolymers containing polyvalent ligand for enhanced interactions with the substrates.
Chitosan is linear, binary heteropolysaccharide and consists of 2-aceta amido-2-deoxy-xcex2-D-glucose (GlcNAc; A-unit) and 2-amino 2-deoxy-xcex2-B-glucose (GIcNAc. D-unit). The active site of lysozyme comprises subsites designated A-F. Specific binding of chitosan sequences to lysozyme begins with binding of the NAG units in the subsite C. Moreover, there is a need to synthesize ligands similar to repeat units of chitosan which will not be hydrolyzed by lysozyme. Moreover natural ligands derived from glucose are susceptible to microbial growth. The copolymers reported here are stable than chitin and chitosan reported earlier.
In our another study entitled xe2x80x9cOligomers and Preparation Thereofxe2x80x9d(Copending application No.), the applicants have shown that the oligomers of NAG in which the NAG groups are juxtaposed to one another, bind more effectively to lysozylne as reflected in values of binding constant (Kb) and the inhibition concentrations I50. In the conventional technique of free radical copolymerization the distribution of monomers along the polymer chain depends upon the values of the monomer reactivity ratios which are determined primarily by the intrinsic structure of the monomer. Consequently the distribution of the NAG units in the copolymers comprising monomers bearing NAG cannot be tailored at will using conventional copolymerization techniques.
To overcome this problem the applicants have devised a novel strategy to ensure that the copolymers prepared using conventional free radical polymerization technique will always contain sequences of NAG units in juxtaposition.
The applicants have further demonstrated that copolymers containing various carbohydrates including NAG units as macromers, bind to lysozyme more strongly as evidenced by values of Kb and inhibit lysozyme more efficiently as evidenced by values of I50. Thus the present invention provides copolymers various carbohydrates including NAG for a biomolecular target and method for preparation thereof.
The approach described to prepare copolymers of various carbohydrates including NAG is simple and can be used to synthesize other macromeric ligands such as sialic acid which bind to influenza virus and rotavirus. Such copolymers may be even used as antiinfective agents both for prevention and treatment of diseases. Moreover, resultant copolymers reported are thernoprecipitating polymers which maybe used for the recovery of biomolecules such as lysozyme and lectins.
The copolymers various carbohydrates including NAG conjugated to polyvalent ligands may also further be used in the treatment of bacterial or viral infections, and are expected not to cause drug resistance.
The approach described herein is a generic one and can be extended to other systems as well for example sialic acid.
The present invention provides methods for the preparation of copolymers containing various carbohydrates including polyvalent N-Acetyl Glucosamine (NAG). The copolymers in this invention are prepared by free radical polymerization method, which is convenient and simple. In the process of copolymerization reported here result in juxtaposition arrangement of polyvalent ligand sequences. Thus, the copolymers provide improved binding and inhibition in term of binding constant (Kb) and inhibition concentration (I50) respect. Copolymers reported here can be used for prevention of viral infections and recoveries of biomolecules.